The number and scope of Organisation for Economic Cooperation and Development (OECD) in vitro test guidelines (TGs) are increasing in an effort to both improve human relevance and replace in vivo animal testing. In vitro test methods being developed for TG use are increasing the use of human based reagents in combination with or replacing animal derived reagents, and demand for human reagents is likely to grow in the near future. There are a range of issues asso­ciated with the ethical use of human reagents, particularly human serum, in the adaptation and development of in vitro TGs, especially to ensure that there is no human exploitation, legal requirements are adhered to, and that the origin of the reagent is assured. To address these concerns, the OECD has instigated a workshop on ethics, sources, availability, and traceability of human based reagents for TG purposes, to be held in March 2019. The focus is to provide guidance on acceptable sources of human serum for use in in vitro TGs, in terms of donor ethics and informed consent regarding commercial use, and quality control for safety and consistent performance, with a view to providing guidance to support the adaptation and/or development of in vitro TGs using human reagents, and to ensure that in reporting the test results to regulators, clearly defined ethical and traceability aspects are adequately addressed, for the Mutual Acceptance of Data principle to be accepted in all OECD member countries. This thought-starter provides a discussion basis to achieve those objectives.

Despite being widely used for basic and preclinical studies in dermatology, available animal models only partly reca­pitulate human skin features, often leading to disappointing outputs when preclinical results are translated to the clinic. Therefore, the need to develop alternative, non-animal models is widely recognized to more closely recapitulate human skin pathophysiology and to address the pressing ethical demand to reduce the number of animals used for research purposes, following the globally accepted 3Rs principle (Replacement, Reduction, and Refinement). Skin is the outermost organ of the body, and is, as such, easily accessible. Different skin cell types can be propagated in vitro, and skin can be reconstructed for therapeutic transplantation as well as for in vitro modelling of pathophysiological conditions. Bio­engineered skin substitutes have been developed and have evolved from elementary to complex systems, more and more closely resembling complete skin architecture and biological responses. In silico analyses take advantage of the huge amount of data already available from human studies to identify and model molecular pathways involved in skin pathophysiology without further animal testing. The present review recapitulates the available non-animal models for der­matological research and sheds light on their prospective technological evolution.

The mouse bioassay for the detection of marine biotoxins in shellfish products is 40 years old and still in use. A full ban or total replacement of this in vivo test has been postponed because of the fear that current chemical-based detection methods could miss a new emerging toxin. In order to fully replace the mouse bioassay, more efforts are needed in the search for functional assays with specific endpoints. Gene expression elicited by diarrheic shellfish poisons (DSP) in Caco-2 cells allowed us to determine three “DSP profiles”, i.e., OA/DTX, AZA-YTX, and PTX profiles. Twelve marker genes were selected to represent the three profiles. qRT-PCR is relatively cheap and easy, and although its multiplex capacity is limited to 5 genes, this was sufficient to show the three expected profiles. The use of the multiplex mag­netic bead-based assay was an even better alternative, allowing the detection of all 12 selected marker genes and 2 reference genes, and resulting in clear profiles with for some genes even higher induction factors than obtained by qRT-PCR. When analyzing blank and contaminated shellfish samples with the multiplex magnetic bead-based assay, the contaminated samples could easily be distinguished from the blank samples, and showed the expected profiles. This work is one step further towards the final replacement of the mouse bioassay. We suggest to combine the neuro-2a bioassay for screening with detection by analytical chemical analyses and with the multiplex magnetic bead-based assay for con­firmation of known and unknown toxins.

Due to the exponentially growing number of substances requiring safety evaluation, efficient prioritisation strategies are needed to identify those of highest concern. To limit unnecessary animal testing, such strategies should respect the 3R principles (Replacement, Reduction, Refinement). In the present study, a strategy based on non-animal approaches was developed to prioritize non-evaluated printed paper and board food contact material (FCM) substances for further in-depth safety evaluation. Within the strategy, focus was put on genotoxicity, a key toxicological endpoint when evaluating safety. By combining in silico predictions with existing in vitro and in vivo genotoxicity data from publicly available literature sources and results from in vitro gene mutation experiments, the 106 study substances could all be assigned to one of the four priority classes (ranging from low to very high concern). Importantly, 19 substances were considered of very high concern due to in vivo genotoxicity. Five of these are furthermore listed as a Substance of Very High Concern (SVHC) by the European Chemicals Agency (ECHA), in addition to demonstrating physicochemical properties linked to a high migration potential as well as oral bioavailability and being used in primary food packaging materials. The current animal-free strategy proved useful for the priority ranking of printed paper and board FCM substances, but it can also be considered to prioritize other substances of emerging concern.

Chemical UV filters are frequently applied as active ingredients in sunscreens to protect from detrimental effects of UV radiation. Regardless, many of these compounds are not well characterized concerning their capacity to counteract UV induced reactive oxygen species (ROS). Intracellular ROS release is an early event upon UV exposure and a crucial trigger of reaction cascades that may provoke adverse effects both in the short- and long-term. We report a strategy to assess the capacity of UV filters (ecamsule, oxybenzone, and menthyl anthranilate) to counteract UVA/UVB stress in the human keratinocyte HaCaT and the wildtype Fibs E6/E7 fibroblast cell lines. The reduction of ROS levels was taken as primary endpoint. The effect of treatment on the cells’ metabolic activity was analyzed as an indicator of viability post-treatment to investigate potential immediate and late (photo)toxicity. Additionally, the compounds’ antioxidative capacity was investigated using an azo-based radical generator. Established antioxidants, quercetin and N-acetylcysteine, were used as controls. Data showed remarkable differences in the mode of action of the chemical UV filters, ranging from protective to pro-oxidative properties, indicating the need for more detailed mode of action-based investigations. Cer­tainly, additional consideration and evaluation will be necessary to further extrapolate these in vitro data for the assessment of in vivo exposure situations. However, the presented approach enables parallel investigations of photoprotective and phototoxic effects of UV filters, and thus can complement and extend existing in vitro testing strategies.

The present study evaluates the applicability of the zebrafish embryotoxicity test (ZET) to assess prenatal develop­mental toxicity (PDT) potency of the DMSO-extracts of 9 petroleum substances (PS), with variable polycyclic aromatic hydrocarbon (PAH) content, and 2 gas-to-liquid (GTL) products, without any PAHs but otherwise similar properties to PS. All PS extracts induced concentration-dependent PDT as quantified in the ZET and this potency is associated with their 3-5 ring PAH content. In contrast and as expected, GTL products did not induce any effect in the ZET. The potencies obtained in the ZET correlated with those previously reported for the embryonic stem cell test (EST) (R2 = 0.61), while the correlation with potencies reported in in vivo studies was higher for the EST (R2 = 0.85) than the ZET (R2 = 0.69). Combining the results of the ZET with those previously reported for the EST (Kamelia et al., 2017), the aryl hydrocarbon (AhR) CALUX assay (Kamelia et al., 2018), and the PAH content, ranked and clustered the test compounds in line with their in vivo potencies and chemical characteristics. Our findings indicate that the ZET does not outperform the EST as a stand-alone assay for testing PDT of PS, but confirm the hypothesis that PAHs are the major inducers of PDT by some PS, while they also indicate that the ZET is a useful addition to a battery of in vitro tests able to predict the in vivo PDT of PS.

Instilling confidence in use of in vitro assays for predictive toxicology requires evaluation of assay performance. Per­formance is typically assessed using reference chemicals – compounds with defined activity against the test system target. However, developing reference chemical lists has historically been very resource-intensive. We developed a semi-automated process for selecting and annotating reference chemicals across many targets in a standardized format and demonstrate the workflow here. A series of required fields defines the potential reference chemical: the in vitro molecular target, pathway, or phenotype affected; and the chemical’s mode (e.g. agonist, antagonist, inhibitor). Activity information was computationally extracted into a database from multiple public sources including non-curated scientific literature and curated chemical-biological databases, resulting in the identification of chemical activity in 2995 biological targets. Sample data from literature sources covering 54 molecular targets ranging from data-poor to data-rich was manually checked for accuracy. Precision rates were 82.7% from curated data sources and 39.5% from automated literature extraction. We applied the final reference chemical lists to evaluating performance of EPA’s ToxCast program in vitro bioassays. The level of support, i.e., the number of independent reports in the database linking a chemical to a target, was found to strongly correlate with likelihood of positive results in the ToxCast assays, although individual assay performance had considerable variation. This overall approach allows rapid development of can­didate reference chemical lists for a wide variety of targets that can facilitate performance evaluation of in vitro assays as a critical step in imparting confidence in alternative approaches.

Experiments with cultured mammalian cells represent an in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used medium supplement worldwide. FCS contains a variable mixture of growth factors and cyto­kines that support cell proliferation. This undefined nature of FCS is a source of experimental variation, undesired immune responses, possible contaminations and, because of the way it is obtained, an ethical concern. Thus, alternative, defined, valid, and reliable medium supplements should be characterized in a large number of experiments. Human platelet lysate (hPL) is increasingly appreciated as an alternative to FCS. Since it is unclear whether cells respond differentially to clin­ically relevant chemotherapeutics inducing replicative stress and DNA damage (hydroxyurea, irinotecan), induction of reactive oxygen species (ROS), the tyrosine kinase inhibitor (TKi) imatinib, and novel epigenetic modifiers belonging to the group of histone deacetylase inhibitors (HDACi), we investigated these issues. Here we show that cancer cells derived from leukemia and colon cancer grow very similarly in culture media supplemented with FCS or outdated hPL. Notably, cells have practically identical proteomes under both culture conditions. Moreover, cells grown with FCS or hPL responded equally to all types of drugs and stress conditions that we tested. In addition, the transfection of blood cells by electroporation can be achieved under both conditions. Furthermore, we reveal that class I HDACs, but not HDAC6, are required for the expression of the pan-leukemic marker WT1 under various culture conditions. Hence, hPL is a moderately priced substitute for FCS in various experimental settings.

Investigative toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, investigative toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adherence to the 3R (Replacement, Reduction, and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, investigative toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of investigative toxicology is to improve pre­clinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded owing to the use of inappropriate animal models. Progress in investigative toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field, a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive, and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an “Investigative Toxicology Think Tank”, which aimed to enhance interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights investigative toxicology’s position by identifying key challenges and perspectives.